Triterpenoid derivatives

ABSTRACT

The present invention relates to the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, in therapy. Preferably, the compound may be used for treating a patient suffering from leukaemia, cancer or other proliferative disorder. A further embodiment relates to the use of a compound of formula (I) in an assay for detecting the phosphorylation and acetylation state of cellular substrates. The present invention also relates to novel compounds of formula (Ia).

[0001] The present invention relates to the therapeutic use and thebiological activity of triterpenoid derivatives. The invention furtherrelates to novel triterpenoid derivatives.

[0002] To date, the prior art has primarily focussed on compounds thatare capable of regulating the cell cycle by virtue of inhibiting cyclindependent kinases (CDKs). Examples of such compounds includebutyrolactone I, flavopiridol, bohemin, olomoucine, roscovitine,purvanalol and indarubicine.

[0003] There is considerable support in the literature for thehypothesis that CDKs and their regulatory proteins play a significantrole in the development of human tumours. Thus, in many tumours atemporal abnormal expression or activity of CDKs has been observed,together with a major deregulation of protein inhibitors (mutations,deletions). This results in the activation of CDKs and consequently indefective regulation of the G1/S transition. Unlike normal cells, tumourcells do not arrest in G1, and since they become independent of growthfactors, they pass the G1 restriction point and enter the S phase veryrapidly.

[0004] In contrast to the prior art, the present invention relates tocompounds which are anti-proliferative, but which are believed tooperate via a mechanism other than CDK inhibition.

[0005] The G₁/S transition of the mammalian cell cycle is tightlyregulated by the retinoblastoma protein (pRb). Retinoblastoma genemutations or deletions predispose individuals to familiar retinoblastomaand other types of cancers. The pRb protein is a docking protein, whichin hypophosphorylated form has the capacity to bind and thus toinactivate S-phase transcription factors such as DP-1 and E2F. However,following phosphorylation by G₁/S cyclin-dependent kiases (CDKs)(CDK4/cyclin D1-D3, CDK6/cyclin D1-D3, CDK2/cyclin A),hyperphosphorylated pRb releases the transcription factors and S phaseis initiated. Within the S phase, the pRb protein phosphorylation ismaintained by the activity of CDK2/cyclin E complexes. Thus,hyperphosphorylation of the pRb protein plays a key role in themolecular pathology of cancer cells with altered CDK activity.

[0006] The present invention relates to the use of triterpenoidcompounds derived from the natural products betulin and betilinic acid(BA) as shown in formula (A). The compounds of the present invention arereferred to hereinafter as bet linines.

[0007] With regard to their biological and therapeutic activity, thecompounds disclosed herein are believed to be of specific benefit in thetreatment of proliferative diseases such as cancers and leukaemias.

[0008] Several of the compounds suitable for use in the presentinvention are already known in the art, for example those disclosed inBer. Dtsch. Chem. Ges. 55, 2332 (1922), Schluze, H. et al; Acta Chem.Scand., B 29, 139 (1975), Suokas E. et al; Collect Czech. Chem. Commun.56, 2936 (1991), Sejbal J. et al; Collect. Czech. Chem. Commun. 64, 329(1999), Klinotová et al; Indian. J. Chem., Sect. B 34, 624 (1995), DindaB. et al, Chem. Listy 91, 1005 (1997), {haeck over (S)}arek J. et al.However, these disclosures do not include any indication as to possiblebiological activity of such compounds.

[0009] A first aspect of the present invention relates to the use of acompound of formula I, or a pharmaceutically acceptable salt thereof, intherapy,

[0010] wherein:

[0011] X¹ is C═O, C═NOR^(1a), CHOR^(1a), CHOCOR^(1a), CHOCOY-Hal,CHOC(O)OR⁹, CHOC(O)OR^(1a), CHOC(O)OR¹⁰, and Hal is Br, Cl, I, F;

[0012] X³ is C═O, CHOR^(1b), CHOCOR^(1b), or X³ and R⁸ together areCHOCOCH₂ and form a spirolactone;

[0013] R¹⁻⁵ are each independently H or lower alkyl;

[0014] R⁶ is H or absent if “a” is a double bond;

[0015] R⁷ is H, COOR^(1c), YOCOR^(1c), COOYOCOR^(1c), YCOOR^(1c);

[0016] R⁸ is H COOR^(1d), YCOOR^(1d), YCOOR¹⁰, YCOHal, COOYOCOR^(1d),CH₂OR^(1d), CH₂COCOR^(1d), COCOCOR^(1d) or R⁷ and R⁸ together are ═CH₂or CH₂OCOCH₂;

[0017] R⁹ is an OH-substituted alkyl group, an ether group or a cyclicether;

[0018] R¹⁰ is lower alkyl substituted by Hal

[0019] “a”, is a double bond or a single bond

[0020] and wherein Y═(CH₂)n

[0021] n=0 to 5;

[0022] R^(1a-1d) are the same or different groups of R¹.

[0023] In a preferred aspect, the invention relates to the use of acompound of formula I, or a pharmaceutically acceptable salt thereof,for treating a patient suffering from leukaemia, cancer or otherproliferative disorder.

[0024] A second aspect of the present invention relates to novelbetulinines of structural formula Ia, or pharmaceutically acceptablesalts, thereof;

[0025] wherein:

[0026] X¹ is C═O, C═NOR^(1a), CHOR^(1a), CHOCOR^(1a), CHOCOY-Hal,CHOC(O)OR⁹, CHOC(O)OR^(1a), CHOC(O)OR¹⁰, and Hal is Br, Cl, I, F;

[0027] X³ is C═O, CHOR^(1b), CHOCOR^(1b), or X³ and X⁸ together areCHOCOCH₂ and form a spirolactone;

[0028] R¹⁻⁵ are each independently H or lower alkyl;

[0029] R⁶ is H or absent if “a” is a double bond;

[0030] R⁷ is H, COOR^(1c), YOCOR^(1c), COOYOCOR^(1c), YCOOR^(1c);

[0031] R⁸ is H, COOR^(1d), YCOOR^(1d), YCOOR¹⁰, YCOHal, COOYOCOR^(1d),CH₂OR^(1d), CH₂COCOR^(1d), COCOCOR^(1d) or R⁷ and R⁸ together are ═CH₂or CH₂OCOCH₂;

[0032] R⁹ is an OH-substituted alkyl group, an ether group or a cyclicether;

[0033] R¹⁰ is lower alkyl substituted by Hal

[0034] “a” is a double bond or a single bond

[0035] and wherein Y═(CH₂)n

[0036] n=0 to 5;

[0037] R^(1a-1d) are the same or different groups of R¹

[0038] with the proviso that

[0039] (i) when X¹ is CHOAc, X³ is C═O, “a” is single bond, R¹⁻⁵ are Me,R⁶ is H;

[0040] when R⁷ is CH₂OAc, R⁸ is other than COOH, CH₂COCOPr^(i),COCOCOPr^(i), CH₂COOH, or CH₂CH₂CO^(i)Pr;

[0041] when R⁷ is CO₂Me, R⁸ is other than CH₂CH₂COCH(CH₃)₂;

[0042] when R⁷is H, R⁸ is other than H, CH₂COOMe, CH₂COOH orCH₂CH₂COPr^(i); and

[0043] (ii) when X¹ is CHOH, X³ is C═O, “a” is single bond, R¹ ⁵ are Me,and R⁶ is H,

[0044] when R⁷ is H, R⁸ is other than H, CH₂COOH, CH₂CH₂COCH(CH₃)₂ orCH₂COOMe;

[0045] when R⁷ is CH₂OAc, R⁸ is other than CH₂COOH;

[0046] or a pharmaceutically acceptable salt thereof.

[0047] As used herein, the term lower alkyl means a linear or branchedchain alllyl group containing from 1 to 6 carbon atoms, includingmethyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] Within the options provided for the groups X¹, X³. and R¹⁻⁸ offormula I, the following options are preferred;

[0049] Preferably,

[0050] X¹ is CHOR^(1a), CHOCOR^(1a) or CHOCOY-Hal; and

[0051] R⁸ is H, COOR^(1d), YCOOR^(1d), COOYOCOR^(1d), CH₂ORld,CH₂COCOR^(1d) or COCOCOR^(1d).

[0052] In a more preferred embodiment, R²⁻⁵ are all methyl and R¹ is asdefined below for the relevant group R^(1a-1d);

[0053] X¹ is —CHOCOCH₂Cl; or

[0054] —CHOR^(1a) or CHOCOR^(1a), wherein R^(1a) is H and methylrespectively;

[0055] X³ is C═O, CHOH or CHOAc;

[0056] R⁷ is H, COOH, COOMe, CH₂OAc, COOYOCOR^(1e) or YCOOR^(1e) where

[0057] Y is CH₂ and R^(1e) is C₁₋₄ alkyl;

[0058] R: is —COOR^(1d), wherein R^(1d) is H or methyl;

[0059] —YCOOR^(1d), wherein Y is CH₂ and R^(1d) is H, methyl or ethyl;

[0060] —COOYOCOR^(1d), wherein Y is CH₂ and R^(1d) is C₁₋₄ alkyl;

[0061] —CH₂OR^(1d), wherein R^(1d) is C₁₋₄ alkyl;

[0062] —CH₂COCOR^(1d) or COCOCOR^(1d), wherein R^(1d) is C₁₋₄ alkyl.

[0063] Of the preferred definitions provided above, it is preferablethat;

[0064] R⁸ is —COOYOCOR^(1d), wherein Y is CH₂ and R^(1d) is methyl orbutyl;

[0065] —CH₂OR^(1d), wherein R^(1d) is methyl or ethyl;

[0066] —CH₂COCOR^(1d) or COCOCOR^(1d), wherein R^(1d) is propyl; and

[0067] R⁷ is COOYOCOR^(1e) or YCOOR^(1e) where Y is CH₂ and R^(1e) ismethyl or butyl.

[0068] In a preferred embodiment, “a” is a single bond and R⁶ is H.

[0069] In a more preferred embodiment of the first aspect of theinvention, the compounds of use are selected from those shown in Table 1below. TABLE 1 No. X¹ R¹ X³ a R²⁻⁵ R⁶ R⁷ R⁸ I.1 CHOAc CH₃ C═O single MeH CH₂OAc CH₂COCOPr¹ I.2 CHOAc CH₃ C═O single Me H CH₂OAc COCOCOPr¹ I.3CHOAc CH₃ C═O single Me H CH₂OAc COOH I.4 CHOAc CH₃ C═O single Me HCH₂OAc COOMe I.5 CHOAc CH₃ C═O single Me H H H I.6 CHOAc CH₃ C═O singleMe H H CH₂COOH I.7 CHOAc CH₃ C═O single Me H H CH₂OEt I.8 CHOAc CH₃ CHOHsingle Me H CH₂OAc COOH I.9 CHOAc CH₃ C═O double Me absent CH₂OAc COOMeI.10 CHOAc CH₃ CHOH single Me H R⁷ and R⁸ together: ═CH₂ I.11 CHOAc CH₃CHOAc single Me H CH₂OAc COOMe I.12 CHOAc CH₃ C═O single Me H COOMeCH₂COCOPr¹ I.13 CHOAc CH₃ C═O single Me H COOMe COOH I.14 CHOAc CH₃ C═Osingle Me H COOMe CH₂COOH I.15 CHOAc CH₃ CHOH single Me H CH₂OAcCOOCH₂OCOBu^(t) I.16 CHOAc CH₃ CHOH single Me H CH₂OAc COOMe I.17 CHOAcCH₃ C═O single Me H CH₂OAc CH₂COOH I.18 CHOAc CH₃ C═O single Me H CH₂OAcCOOCH₂OCOBu^(t) I.19 CHOAc CH₃ C═O single Me H CH₂OAc COOCH₂OCOMe I.20CHOAc CH₃ C═O double Me absent CH₂OAc COOCH₂OCOBu^(t) I.21 CHOAc CH₃ C═Odouble Me absent CH₂OAc COOCH₂OCOMe I.22 CHOAc CH₃ C═O double Me absentCH₂OAc COOH I.24 CHOAc CH₃ C═O single Me H R⁷ and R⁸ together: ═CH₂ I.26CHOH H C═O single Me H CH₂OAc COOH I.27 CHOH H C═O double Me absentCH₂OAc COOMe I.28 CHOAc CH₃ C═O single Me H COOCH₂OCOMe CH₂COCOPr¹ I.29CHOAc CH₃ C═O single Me H COOCH₂OCOBu^(t) CH₂COCOPr¹ I.30 CHOAc CH₃ C═Osingle Me H from R⁷ to R⁸: CH₂OC(O)CH₂ I.31 CHOAc CH₃ CHOAc single Me HCH₂OAc COOH I.32 CHOAc CH₃ CHOAc single Me H CH₂OAc COOCH₂OCOBu^(t) I.33CHOAc CH₃ CHOH single Me H CH₂OAc COOMe I.34 CHOAc CH₃ CHOAc single Me HCH₂OAc COOMe I.35 CHOAc CH₃ CHOH single Me H CH₂OAc COOCH₂OCOBu^(t) I.36CHOAc CH₃ CHOAc single Me H CH₂OAc COOCH₂OCOBu^(t) I.37 CHOAc CH₃ C═Osingle Me H CH₂OAc COOMe I.38 CHOAc CH₃ C═O single Me H H CH₂COOMe I.39CHOAc CH₃ C═O single Me H COOMe CH₂COOMe I.40 CHOAc CH₃ C═O single Me HCOOCH₂OCOBu^(t) CH₂COOMe I.41 CHOAc CH₃ C═O single Me H COOCH₂OCOMeCH₂COOMe I.42 CHOAc CH₃ CHOH single Me H R⁷ and R⁸ together: ═CH₂ I.43CHOAc CH₃ CHOH single Me H H H I.44 CHOCOCH₂Cl CH₃ C═O single Me HCH₂OAc COOH I.45 CHOAc CH₃ CHOH single Me H CH₂OAc COOH I.46 CHOAc CH₃CHOAc single Me H CH₂OAc COOH I.47 CHOAc CH₃ CHOAc single Me H R⁷ and R⁸together: ═CH₂ I.48 CHOAc CH₃ CHOAc single Me H H H I.49 CHOH CH₃ C═Osingle Me H H CH₂COOH I.50 CHOH CH₃ C═O single Me H H CH₂COOMe I.51 CHOHCH₃ CHOH single Me H H CH₂COOMe I.52 CHOAc CH₃ CHOH single Me H HCH₂COOMe I.53 CHOH CH₃ CHOH single Me H CH₂COOMe H I.54 CHOAc CH₃ CHOAcsingle Me H CH₂COOMe H I.55 CHOAc CH₃ {circle over (1)} single Me H H{circle over (1)} I.56 CHOH CH₃ {circle over (1)} single Me H H {circleover (1)} I.57 CHOH CH₃ C═O single Me H CH₂OAc COOH I.58 CHOAc CH₃ C═Osingle Me H CH₂OAc COF

[0070] In a most preferred embodiment, the compound is selected from;

[0071] In respect of the second aspect of the invention, the preferredembodiments regarding the compounds are identical to those given abovefor the first aspect with application of the proviso of formula Ia.

[0072] The most preferred compounds of the second aspect are those inTable 1a below. TABLE 1a No. X¹ R¹ X³ a R²⁻⁵ R⁶ R⁷ R⁸ I.4 CHOAc CH₃ C═Osingle Me H CH₂OAc COOMe I.7 CHOAc CH₃ C═O single Me H H CH₂OEt I.8CHOAc CH₃ CHOH single Me H CH₂OAc COOH I.9 CHOAc CH₃ C═O double Meabsent CH₂OAc COOMe I.10 CHOAc CH₃ CHOH single Me H R⁷ and R⁸ together:═CH₂ I.11 CHOAc CH₃ CHOAc single Me H CH₂OAc COOMe I.12 CHOAc CH₃ C═Osingle Me H COOMe CH₂COCOPr¹ I.13 CHOAc CH₃ C═O single Me H COOMe COOHI.14 CHOAc CH₃ C═O single Me H COOMe CH₂COOH I.15 CHOAc CH₃ CHOH singleMe H CH₂OAc COOCH₂OCOBu^(t) I.16 CHOAc CH₃ CHOH single Me H CH₂OAc COOMeI.18 CHOAc CH₃ C═O single Me H CH₂OAc COOCH₂OCOBu^(t) I.19 CHOAc CH₃ C═Osingle Me H CH₂OAc COOCH₂OCOMe I.20 CHOAc CH₃ C═O double Me absentCH₂OAc COOCH₂OCOBu^(t) I.21 CHOAc CH₃ C═O double Me absent CH₂OAcCOOCH₂OCOMe I.22 CHOAc CH₃ C═O double Me absent CH₂OAc COOH I.24 CHOAcCH₃ C═O single Me H R⁷ and R⁸ together: ═CH₂ I.26 CHOH H C═O single Me HCH₂OAc COOH I.27 CHOH H C═O double Me absent CH₂OAc COOMe I.28 CHOAc CH₃C═O single Me H COOCH₂OCOMe CH₂COCOPr¹ I.29 CHOAc CH₃ C═O single Me HCOOCH₂OCOBu^(t) CH₂COCOPr¹ I.30 CHOAc CH₃ C═O single Me H from R⁷ to R⁸:CH₂OC(O)CH₂ I.31 CHOAc CH₃ CHOAc single Me H CH₂OAc COOH I.32 CHOAc CH₃CHOAc single Me H CH₂OAc COOCH₂OCOBu^(t) I.33 CHOAc CH₃ CHOH single Me HCH₂OAc COOMe I.34 CHOAc CH₃ CHOAc single Me H CH₂OAc COOMe I.35 CHOAcCH₃ CHOH single Me H CH₂OAc COOCH₂OCOBu^(t) I.36 CHOAc CH₃ CHOAc singleMe H CH₂OAc COOCH₂OCOBu^(t) I.37 CHOAc CH₃ C═O single Me H CH₂OAc COOMeI.39 CHOAc CH₃ C═O single Me H COOMe CH₂COOMe I.40 CHOAc CH₃ C═O singleMe H COOCH₂OCOBu^(t) CH₂COOMe I.41 CHOAc CH₃ C═O single Me H COOCH₂OCOMeCH₂COOMe I.42 CHOAc CH₃ CHOH single Me H R⁷ and R⁸ together: ═CH₂ I.43CHOAc CH₃ CHOH single Me H H H I.44 CHOCOCH₂Cl CH₃ C═O single Me HCH₂OAc COOH I.45 CHOAc CH₃ CHOH single Me H CH₂OAc COOH I.46 CHOAc CH₃CHOAc single Me H CH₂OAc COOH I.47 CHOAc CH₃ CHOAc single Me H R⁷ and R⁸together: ═CH₂ I.48 CHOAc CH₃ CHOAc single Me H H H I.51 CHOH CH₃ CHOHsingle Me H H CH₂COOMe I.52 CHOAc CH₃ CHOH single Me H H CH₂COOMe I.53CHOH CH₃ CHOH single Me H CH₂COOMe H I.54 CHOAc CH₃ CHOAc single Me HCH₂COOMe H I.55 CHOAc CH₃ {circle over (1)} single Me H H {circle over(1)} I.56 CHOH CH₃ {circle over (1)} single Me H H {circle over (1)}I.57 CHOH CH₃ C═O single Me H CH₂OAc COOH I.58 CHOAc CH₃ C═O single Me HCH₂OAc COF

[0073] In respect of the invention as a whole, it is preferable that theproliferative disorder is cancer or leukaemia. In one embodiment, thecancer or leukaemia is p53, hormone and multidrug resistanceindependent. In another embodiment, the cancer or leukaemia isindependent of Rb status.

[0074] More specifically, the present invention relates to a method oftreating patients suffering from cancer by administering therapeuticallyeffective amounts of a compound of formula I or pharmaceuticallyacceptable salts or esters thereof.

[0075] Without wishing to be bound by theory, preliminary studiessuggest that rather than influencing the activity of cyclin dependentkinases, the compounds of the present invention appear to operate via analternative mechanism. In particular, it is believed that thebetulinines of the present invention may inhibit cell proliferation andinduce cancer cell death in a manner which involves mainlypost-translational modifications, namely the phosphorylation, of a keyregulatory protein involved in cellular proliferation. Morespecifically, it is believed that the betulinines of the inventioneffect a change in the phosphorylation state of the Rb protein. Such amechanism may be advantageous as it is thought that the compounds of thepresent invention may be capable of inhibiting cell proliferation inproliferating tumour tissue, but not in healthy tissue.

[0076] Thus, in a further embodiment the present invention relates to amethod of treating a cancerous or leukaemic proliferative diseasethrough effecting a change in the pRb protein phosphorylation state bythe administration of a therapeutically effective amount of a compoundof formula I or pharmaceutically acceptable salts or esters thereof.

[0077] The compounds of the present invention are also capable ofinducing apoptosis (programmed cell death) in proliferative cells. Thus,in an additional embodiment, the present invention relates to a methodof inducing cell death in proliferative cells comprising admi iseig atherapeutically effective amount of a compound of formula I orpharmaceutically acceptable salts or esters thereof.

[0078] A further aspect of the present invention relates to use ofbetulinines of formula I as research chemicals and as compounds forclinical and/or laboratory diagnostics. More particularly, the inventionrelates to the use of betulinines as research chemicals for studying thephosphorylation/de-phosphorylation processes of cellular substrates,cellular proliferation, purification of target molecules, and/or cellcycle studies.

[0079] The present invention therefore further relates to the use of acompound of formula I in the preparation of a medicament for use in thetreatment of a proliferative disease.

[0080] As used herein the phrase “preparation of a medicament” includesthe use of a compound of formula I directly as the medicament inaddition to its use in a screening programme for the identification offurther anti-proliferative agents or in any stage of the manufacture ofsuch a medicament

[0081] Such a screening programme may for example include an assay fordetermining the phosphorylation state of cellular substrates anddetermining whether a candidate substance is capable of mimicking theactivity of a betulinine of formula I.

[0082] Thus, in a further embodiment, the invention relates to the useof a compound of formula I or a pharmaceutically acceptable salt,crystal form, complex, hydrate, or hydrolysable ester thereof, in anassay for determining the phosphorylation state of cellular substrates,and optionally in the identification of candidate compounds that act ina similar manner.

[0083] Preferably, the cellular substrate, the phosphorylation state ofwhich is being assayed is Rb protein.

[0084] The compounds of the fir&t and second aspects of the presentinvention can be present as salts or esters, in particularpharmaceutically acceptable salts or esters.

[0085] Pharmaceutically acceptable salts of the product of the inventioninclude suitable acid addition or base salts thereof A review ofsuitable pharmaceutical salts may be found in Berge et al, J Pharm Sci,66, 1-19 (1977). Salts are formed, for example with strong inorganicacids such as mineral acids, e.g. sulphuric acid, phosphoric acid orhydrohalic acids; with strong organic carboxylic acids, such asalkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted orsubstituted (e.g., by halogen), such as acetic acid; with saturated orunsaturated dicarboxylic acids, for example oxalic, malonic, succinic,maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylicacids, for example ascorbic, glycolic, lactic, malic, tartaric or citricacid; with amino acids, for example aspartic or glutamic acid; withbenzoic acid; or with organic sulfonic acids, such as (C₁-C₄)alkyl- oraryl-sulfonic acids which are unsubstituted or substituted (for example,by a halogen) such as methane- or p-toluene sulfonic acid.

[0086] Esters are formed either using organic acids oralcohols/hydroxides, depending on the functional group being esterified.Organic acids include carboxylic acids, such as alkanecarboxylic acidsof 1 to 12 carbon atoms which are unsubstituted or substituted (e.g., byhalogen), such as acetic acid; with saturated or unsaturateddicarboxylic acid, for example oxalic, malonic, succinic, maleic,fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, forexample ascorbic, glycolic, lactic, malic, tartaric or citric acid; withamino acids, for example aspartic or glutamic acid; with benzoic acid;or with organic sulfonic acids, such as (C₁-C₄)-alkyl- or aryl-sulfonicacids which are unsubstituted or substituted (for example, by a halogen)such as methane- or p-toluene sulfonic acid. Suitable hydroxides includeinorganic hydroxides, such as sodium hydroxide, potassium hydroxide,calcium hydroxide, aluminium hydroxide. Alcohols include alkanealcoholsof 1-12 carbon atoms which may be unsubstituted or substituted, e.g. bya halogen).

[0087] In all aspects of the present invention previously discussed, theinvention includes, where appropriate all enantiomers and tautomers ofcompounds of formula I or Ia. The man skilled in the art will recognisecompounds that possess optical properties (one or more chiral carbonatoms) or tautomeric characteristics. The corresponding enantiomersand/or tautomers may be isolated/prepared by methods known in the art

[0088] The invention furthermore relates to the compounds of, or of use,in the present invention in their various crystalline forms, polymorphicforms and (an)hydrous forms. It is well established within thepharmaceutical industry that chemical compounds may be isolated in anyof such forms by slightly varying the method of purification and orisolation from the solvents used in the synthetic preparation of suchcompounds.

[0089] The invention further includes the compounds of, or of use, inthe present invention in prodrug form. Such prodrugs are generallycompounds of formula I or Ia wherein one or more appropriate groups havebeen modified such that the modification is reversed upon administrationto a human or mammalian subject. Such reversion is usually performed byan enzyme naturally present in such subject, though it is possible for asecond agent to be administered together with such a prodrug in order toperform the reversion in vivo. Examples of such modifications includeesters (for example, any of those described above), wherein thereversion may be carried out be an esterase etc. Other such systems willbe well known to those skilled in the art.

[0090] The present invention also encompasses pharmaceuticalcompositions comprising the compounds of the invention. In this regard,and in particular for human therapy, even though the compounds of thepresent invention (including their pharmaceutically acceptable salts,esters and pharmaceutically acceptable solvates) can be administeredalone, they will generally be administered in admixture with apharmaceutical carrier, excipient or diluent selected with regard to theintended route of administration and standard pharmaceutical practice.

[0091] Thus, the present invention also relates to pharmaceuticalcompositions comprising betulinines or pharmaceutically acceptable saltsor esters thereof, together with at least one pharmaceuticallyacceptable excipient, diluent or carrier.

[0092] By way of example, in the pharmaceutical compositions of thepresent invention, the compounds of the invention may be admixed withany suitable binder(s), lubricant(s), suspending agent(s), coatingagent(s), and/or solubilising agent(s). Examples of such suitableexcipients for the various different forms of pharmaceuticalcompositions described herein may be found in the “Handbook ofPharmaceutical Excipients, 2^(nd) Edition, (1994), Edited by A Wade andP J Weller.

[0093] The pharmaceutical compositions of the present invention may beadapted for oral, rectal, vaginal, parenteral, intramuscular,intraperitoneal, intraarterial, intrathecal, intrabronchial,subcutaneous, intradermal, intravenous, nasal, buccal or sublingualroutes of administration.

[0094] For oral administration, particular use is made of compressedtablets, pills, tablets, gellules, drops, and capsules. Preferably,these compositions contain from 1 to 250 mg and more preferably from10-100 mg, of active ingredient per dose.

[0095] Other forms of administration comprise solutions or emulsionswhich may be injected intravenously, intraarterially, intrathecally,subcutaneously, intradermally, intraperitoneally or intramuscularly, andwhich are prepared from sterile or sterilisable solutions. Thepharmaceutical compositions of the present invention may also be in formof suppositories, pessaries, suspensions, emulsions, lotions, ointments,creams, gels, sprays, solutions or dusting powders.

[0096] An alternative means of transdermal administration is by use of askin patch. For example, the active ingredient can be incorporated intoa cream consisting of an aqueous emulsion of polyethylene glycols orliquid paraffin. The active ingredient can also be incorporated, at aconcentration of between 1 and 10% by weight, into an ointmentconsisting of a white wax or white soft paraffin base together with suchstabilisers and preservatives as may be required.

[0097] Injectable forms may contain between 10-1000 mg, preferablybetween 10-250 mg, of active ingredient per dose.

[0098] Compositions may be formulated in unit dosage form, i.e., in theform of discrete portions containing a unit dose, or a multiple orsub-unit of a unit dose.

[0099] A person of ordinary skill in the art can easily determine anappropriate dose of one of the instant compositions to administer to asubject without undue experimentation. Typically, a physician willdetermine the actual dosage which will be most suitable for anindividual patient and it will vary with the age, weight and response ofthe particular patient The dosages disclosed herein are exemplary of theaverage case. There can of course be individual instances where higheror lower dosage ranges are merited, and such are within the scope ofthis invention.

[0100] In an exemplary embodiment, one or more doses of 10 to 150 mg/daywill be administered to the patient for the treatment of malignancy.

[0101] The invention further relates to methods of chemical synthesis ofthe above described compounds.

[0102] In one embodiment, the invention relates to a process forpreparing compounds of formula I as defined above, comprising:

[0103] (i) oxidising a compound of formula Ib to a compound of formulaIc;

[0104] (ii) reducing said compound of formula Ic to form a compound offormula Id, and optionally

[0105] (iii) converting said compound of formula Id to a compound offormula I wherein “a” is a double bond.

[0106] In a preferred embodiment, the compound of formula Ib is oxidisedto Ic by treating sequentially with selenium dioxide, peroxyacetic acidand ruthenium tetroxide.

[0107] In a more preferred embodiment, the reduction of Ic to Id is astereoselective reduction.

[0108] Even more preferably, the compound of formula Ic is reduced to Idby treating with sodium borohydride in the presence of a cerium (III)salt.

[0109] In a preferred aspect, step (iii) comprises esterifying acompound of formula Id wherein R^(1d) is H, oxidising anddehydrogenating.

[0110] The preparation of the compounds of the present invention will bediscussed in greater detail below, with specific reference to thepreferred embodiments. The man skilled in the relevant art would be ableto prepare other compounds of the invention by selection of theappropriate reagents.

[0111] The following scheme illustrates the synthesis of compounds offormula I where X¹ is CHOAc, X² is CH₂, X³ is C═O, R¹⁻⁵ are methyl, R⁶is H or absent (when “a” is a double bond), R⁷ is CH₂OAc, and R⁸ isCOOH, COOCH₂OCOMe, or COOCH₂OCOC(CH₃)₃.

[0112] Conditions: a, Acetylation with acetic anhydride in the presenceof base (e.g. pyridine); b, isomerisation of double bond by treatmentwith hydrogen bromide in acetic acid; c, oxidation (e.g. with sodiumdichromate); d, oxidation (e.g with selenium dioxide); e, oxidation(e.g. with peroxyacetic acid); f, fission of double bond (e.g. withruthenium tetroxide); g, stereoselective reduction (e.g. with sodiumborohydride in the presence of a cerium(III) salt); h, esterificationwith chloromethyl pivalate (POM-Cl) in the presence of base (e.g.1,8-diazabicyclo[5,4,0]undec-7-ene (DBU)); i, esterification withbromomethyl acetate (AcM-Br) in the presence of base (e.g. DBU); j,dehydrogenation (e.g. with selenium dioxide); k, hydrolysis (e.g. withbis(tributyltin)oxide).

[0113] The scheme below illustrates the synthesis of compound I.58 offormula I where X¹ is CHOAc, X³ is C═O, R¹-R⁵ are methyls, R⁶ is H, R⁷is CH₂OAc, R⁸ is COF.

[0114] Conditions: 1, reaction with diethylaminosulphur trifluoride.

[0115] The present invention is also described with reference to theaccompanying figures wherein:

[0116]FIG. 1A shows an electron micrograph of a normal, untreated AS49lung cancer cell attached to coverslip with a number of well formedpseudopods and fine structure of cytoplasmatic membrane.

[0117]FIG. 1B shows an electron micrograph of betulinic acid treatedAS49 lung cancer cells. The left cell is attached to the coverslip andexhibits normal morphology with fine cytoplasmatic membrane structure,while the right cell already shows cytoplasmatic membrane blebbing, anearly sign of apoptosis.

[0118]FIG. 1C shows an electron micrograph of I.3 treated A549 lungcancer cells. This illustrates a typical apoptotic tumour cell withextensive cytoplasmatic membrane blebbing, detachment and formation ofapoptotic bodies.

[0119]FIG. 1D is a magnification of FIG. 1C, showing details ofapoptotic body formation in I.3 treated cells.

[0120]FIG. 2 shows the anticancer activity of betuliine I.3 inxenotrasplantated human SK-N-AS neuroblastoma

[0121]FIG. 3 shows the results of SDS-PAGE electrophoresis of totalcellular proteins. In particular, the gel shows that betulinine I.3, butnot betulinic acid (BA) induces rapid dephosphorylation of Rb protein.

[0122]FIG. 4A shows a cell cycle analysis of cells treated with I.3 andpaclitaxel as a control.

[0123]FIG. 4B shows the induction of apoptosis in cells treated with I.3or paclitaxel as a control.

[0124] More detailed reference to the above figures may be found in theaccompanying examples.

[0125] This invention is further illustrated by the following examples,which should not be construed as further limiting.

EXAMPLES

[0126] General

[0127] The chemical shift values (δ-scale, ppm) and coupling constants(J, Hz) in the ¹H and ¹³C NMR spectra were obtained using a VarianUNITY-INOVA 400 FT spectrometer (¹H at 400 MHz and ¹³C at 100.6 MHz) indeuterochloroform with tetramethylsilane (for ¹H NMR data δ=0 ppm) as aninternal standard. For the ¹³C NMR data δ(CDCl₃)=77.00 ppm. The valuefor a multiplet, either defined (doublet (d), triplet (t), quartet (q),septet (sept) or not (m) at the approximate mid point is given unless arange is quoted (s=singlet, b=broad)).

[0128] Electron impact mass spectra (EIMS) were measured on an INCOS 50instrument ionising electron energy 75 eV, ion source temperature 150°C. EIMS was used to determine molecular weights, M⁺ corresponding to themolecular ion.

[0129] Ether is diethylether. THF and dioxane were dried over sodium.Acetic acid was purified before use by chromium trioxide treatment anddistillation. Reactions were run at room temperature unless otherwisestated. The reaction progress was monitored by thin layer chromatography(TLC) on silicagel 60 G (Merck, detection by spraying with 10% sulphuricacid and heating). The work-up procedure involves dilution withspecified solvent (otherwise the organic reaction solvent), extractionwith water and then brine or sodium hydrogencarbonate, drying overanhydrous magnesium sulphate, and evaporation under vacuum to give aresidue.

Example 1 Lup-20(29)-ene-3β,28-diyl diacetate

[0130] Crude betuline (500 g) was dissolved in a mixture of 250 mlpyridine and 250 ml acetic anhydride. The mixture was then refluxed forhalf an hour. After cooling, the resulting crystals were filtered offand washed with acetic acid, ethanol and water. A solution of crudelup-20(29)-ene-3β,28-diyl diacetate (400 g) in chloroform was filteredthrough a column of alumina, and the column was washed with chloroform.The filtrate was then evaporated under reduced pressure. The residue wascrystallized from chloroform/methanol to obtain 250 g of the titlecompound which according to TLC contained traces of lupeol acetate.After re-crystallization from chloroform/methanol, the yield of purecompound was 239 g, mp. 222-223° C, [α]_(D)+22° (c 0.4; CHCl₃). [SchulzeH., Pieroh K: Ber. Dtsch Chem. Ges. 55,2332 (1922)].

[0131] The ¹H NMR spectrum of the title compound is as follows: 0.84 s,0.84 s, 0.85 s, 0.97 s, 1.03 s, 1.68, 6×3H (6×CH₃); 2.04 s, 3 H, 2.07 s,3 H (2×OAc); 2.44 ddd, 1 H (J′=11.4, J″=10.9, J′″=0.7, H-19); 3.85 d, 1H (J=11.1, H-28a); 4.25 dd, 1 H, (J′=11.1,J″=1.4, H-28b);4.47 m, 1 H(H-3a); 4.59 m, 1 H (Σ J=3.4, H-29E); 4.69 m, 1 H (Σ J=2.1, H-29Z).

Example 2 Lup-18-ene-3β,28-diyl diacetate

[0132] A solution of hydrogen bromide in acetic acid (38%, 1.4 l) wasadded to a solution of lup-20(29)-ene-3β,28-diyl diacetate (100 g, 190mmol) in a mixture of benzene, acetic acid and acetic anhydride(11:0.51:50 ml). The reaction mixture was refluxed until the reactionwas completed (TLC was developed in hexane/ether mixture). Aftercooling, the reaction mixture was poured into ice cold water (3:1) andextracted with benzene (3×0.51 l). The combined organic phases werewashed with NaHCO₃ aqueous solution, NaHSO₃ solution and water and driedover magnesium sulphate. Usual working up procedure gave 90 g of darkbrown residue. The dry powder was extracted in a Soxhlet extractor withacetone until it turned white. After drying in the air, the product wascrystallized from butanone. The yield of the title compound was 74 g(74%), mp. 215-216° C., [α]_(D) +15° (c 0.45; CHCl₃). [Suokas E., HaseT.: Acta Chem. Scand., B 29, 139 (1975)].

[0133] The ¹H NMR spectrum of the title compound is as follows: 0.84 s,0.85 s, 0.89 s, 0.90 s, 0.91 d, 3 H (J=6.8), 0.99 d, 3 H (J=6.8), 1.06s, 7×3H (7×CH₃); 2.04 s, 3 H, 2.05 s, 3 H (2×OAc); 2.25 m, 2 H (Σ J˜15);2.43 m, 1 H (Σ J˜15); 3.14 sept., 1 H (J=7, H-20); 3.98 d, 1 H (J=10.8,H-28a); 4.03 d, 1 H (J=10.8, H-28b); 4.49 m, 1 H (H-3α).

Example 3 21-oxo-lup-18-ene-3β,28-diyl diacetate

[0134] Lup-18-ene-3β,28-diyl diacetate (50 g; 95 mmol), sodiumdichromate (22.5 g; 75.5 mmol) and sodium acetate (5 g) were dissolvedin a mixture of benzene and acetic acid (0.7 l, 0.3 l). The reactionmixture was allowed to stand until the reaction was completed (TLC wasdeveloped in hexane/ether). After dilution with an excess of water, themixture was extracted with benzene (3×300 ml). After usual working upprocedure the title compound was obtained (45 g, 87%) as a pale-yellowcrystaline foam which was used in the next step without furtherpurification (see Example 4). Pure title compound has m.p. 205-206° C.,[α]_(D) −35° (c 0.49; CHCl₃). Another way to the title compound isdescribed in Sejbal J., Klinot J., Bude{haeck over (s)}ínský M., ProtivaJ.: Collect. Czech Chem. Commun. 56,2936 (1991).

[0135] The ¹H NMR spectrum of the title compound is as follows: 0.85 s,0.86 s, 0.93 s, 0.94 s, 1.16 s, 1.17 d (J=7.1), 1.21 d (J=7.1), 7×3H(7×CH₃); 2.00 s, 3 H, 2.05 s, 3 H (2×OAc); 2.39 d, 1 H (J=18.5, H-22);2.87 dd, 1 H (J′=11.9, J″=4.1, H-13β); 3.18 sept, 1 H (J=6;6, H-20);4.06 d, 1 H (J=10.9, H-28a); 4.34 d, 1 H (J=10.9, H-28b); 4.49 m, 1 H(J˜7, H-3α).

[0136] The following compounds were prepared by the above-mentionedprocedure:

[0137] (pivaloyloxy)methyl3β,28-diacetoxy-18-oxo-19,20,21,29,30-pentanorlupan-22-oate

[0138] acetoxymethyl3β,28diacetoxy-18-oxo-19,20,21,29,30-pentanorlupan-22-oate

Example 4 21,22-dioxolup-18-ene-3β,28-diyl diacetate

[0139] A solution of crude 21-oxolup-18-ene-3β,28-diyl diacetate (40 g;74 mmol; containing about 85% of 21-oxo-lup-18-ene-3β,28-diyl diacetate)and selenium dioxide (160 g; 1.44 mol) in a mixture of dioxane (0.8 l)and acetic acid (0.4 l) was refluxed until the reaction was completed(TLC was developed in benzene/ether).

[0140] After cooling, the precipitated selenium was removed byfiltration and the filtrate was slowly poured into a vigorously stirredexcess of water. The red-orange precipitate was filtered off underreduced pressure, carefully washed with water and dried in the air. Drycrude 21,22dioxo-lup-18-ene-3β,28-diyl diacetate was dissolved inchloroform and the solution was filtered through a column of alumina,the column was then washed with chloroform, and the filtrate wasevaporated under reduced pressure. The residue was crystallized frommethyl acetate to give 28.9 g (82%) of the title compound as pale-orangeneedles, mp. 267-270° C., [α]_(D) −127° (c 0.32; CHCl₃). Another way tothe title compound is described in Klinotová E., Cermáková J., RejzekM., Krecek V., Sejbal J., Ol{haeck over (s)}ovský P., Klinot J.:Collect. Czech Chem. Commun. 64, 329 (1999).

[0141] The ¹H NMR spectrum of the title compound is as follows: 0.85 s,0.86 s, 0.94 s, 0.97 s, 1.18 s, 1.24 d (J=7.2), 1.26 d (J=7.2), 7×3 H(7×CH₃); 1.93 s, 3 H, 2.06 s, 3 H (2×OAc); 3.12 dd, 1 H (J′=12.5,J″=3.8, H-13β), 3.36 sept., 1 H (J=7.0, H-20); 4.02 d, 1 H (J=11.1,H-28a); 4.49 dd, 1 H (J′=10.2, J″=6.0, H-3α); 4.84 d, 1 H (J=11.1,H-28b).

[0142] The following compounds were prepared by the above-mentionedprocedure:

[0143] methyl 3β-acetoxy-21,22dioxolup-18-en-28-oate

[0144] acetoxymethyl 3β-acetoxy-21,22-dioxolup-18-en-28-oate

[0145] (pivaloyloxy)methyl 3β-acetoxy-21,22-dioxolup-18-en-28-oate

[0146] acetoxymethyl3β,28-diacetoxy-18-oxo-19,20,21,29,30-pentanorlup-12-en-22-oate

[0147] (pivaloyloxy)methyl3β,28diacetoxy-18-oxo-19,20,21,29,30-pentanorlup-12-en-22-oate

[0148] 3β-hydroxy-30-oxolup-20(29)-en-28-oic acid [Dinda B., Hajra A.K., Das S. K., Chel G., Chakraborty R., Ranu B. C.: Indian. J. Chem.,Sect. B 34, 624 (1995)). acetoxymethyl3β-hydroxy-30-oxolup-20(29)-en-28-oate (pivaloyloxy)methyl3β-hydroxy-30-oxolup-20(29)-en-28-oate

Example 5 Anhydride of 3β,28-diacetoxy-21,22-secolup-18-ene-21,22-dioicacid

[0149] A solution of 21,22-dioxolup-18-ene-3β,28-diyl diacetate (25 g;45 mmol) and peroxyacetic acid (0.6 l; 32%) in chloroform (0.25 l) wasvigorously stirred until the reaction was completed (TLC was developedin hexane/ether). The colourless reaction mixture was diluted with coldwater and extracted with chloroform (3×200 ml). The combined organicphases were washed with 5% aqueous solution of potassium iodide (400ml), saturated aqueous solution of sodium sulphite (200 ml) and brine(2×200 ml), dried and evaporated. The resulting pale-yellow oil wascrystallized from chloroform/methanol to give 20.7 g (80.5%) of thetitle compound as small white crystals, m.p. 306-309° C., [α]_(D) +88°(c 0.45; CHCl₃). Another way to the title compound is described inSejbal J., Kilnot J., Bude{haeck over (s)}ínský M., Protiva J.: Collect.Czech. Chem. Commun. 56, 2936 (1991).

[0150] The ¹H NMR spectrum of the title compound is as follows: 0.85 s,0.85 s, 0.90 s, 0.91 s, 1.11 s, 1.14 d (J=7), 1.31 d (J=7), 7×3H(7×CH₃); 2.01 s, 3 H, 2.05 s, 3 H (2×OAc); 2.53 dt, 1 H (J′=14.4,J″=J′″=3.5); 2.72 dd, 1 H (J′3.1, J″=12.3 ); 3.26 sept, 1 H (H-20, J=7);3.90 d, 1 H, 4.54 d, 1 H (2×H-28, J=11.0); 4.47 m, 1 H (H-3α).

[0151] The following compounds were prepared by the above-mentionedprocedure:3β,28-diacetoxy-18-oxo-18,19-seco-19,20,29,30-tetranorlupan-21-oic acid[{haeck over (S)}arek J., Klinot J., Klinotová E., Sejbal J.: Chem.Listy 91, 1005 (1997)],3β,28-diacetoxy-18-oxo-19,20,21,29,30-pentanorlupan-22-oic acid.

Example 6 3β,28-diacetoxy-18-oxo-19,20,21,29,30-pentanorlupan-22-oicacid

[0152] The anhydride of 3β,28-diacetoxy-21,22-secolup-18-ene-21,22-dioicacid (20 g; 35.1 mmol) in ethyl acetate (1 l) was added to a mixture ofruthenium dioxide (400 mg; 4 mmol), sodium metaperiodate (60 g; 280.3mmol), water (200 ml) and trifluoroacetic acid (20 ml), and the mixturewas vigorously stirred. After the reaction was completed, ethanol wasadded, the mixture was filtered, and the organic layer was filteredthrough a short silica gel column. The column was then washed with ethylacetate, the filtrate was evaporated under the reduced pressure andresidue was washed with ether and crystallized from a mixture ofdichloromethane/ether. The yield of the title compound was 10.6 g (61%),m.p. 137-140° C., [α]_(D) +40° (c 0.37; CHCl₃). [{haeck over (S)}arekJ., Klinot J., Klinotová E., Sejbal J.: Chem. Listy 91, 1005 (1997)].

[0153] The ¹³C NMR spectrum of the title compound is as follows:

[0154] 213.3, 174.2, 171.1, 170.5, 80.7, 65.4, 58.8, 55.4, 50.6, 50.1,46.8, 41.1, 38.5, 37.8, 37.1, 33.9, 28.4, 27.9, 26.8, 23.5, 21.8, 21.2,20.6, 19.6, 18.1, 16.7, 16.5, 16.2, 16.0.

[0155] The following compounds were prepared by the above-mentionedprocedure: 18,19,21-trioxo-18,19-secolupane-3β,28-diyl diacetate [{haeckover (S)}arek J., Klinot J., Klinotová E., Sejbal J.: Chem. Listy 91,1005 (1997)]. 18,19,21,22-tetraoxo-18,19-secolupane-30,28-diyl diacetate3β-acetoxy-21-oxolup-18-en-28-oic acid

Example 7 3β,28-diacetoxy-18-hydroxy-19,20,21,29,30-pentanorlupan-22-oicacid

[0156] To a solution of3β,28-diacetoxy-18-oxo-19,20,21,29,30-pentanorlupane-22-oic acid (10 g;19.8 mmol) in 500 ml TEF, a solution of cerium chloride (200 ml; 1 M)and sodium borohydride (2 g; 53 mmol ) was added. The reaction mixturewas stirred for one hour. The mixture was poured into an excess of 1%aqueous solution of hydrochloric acid and extracted with chloroform(3×300 ml). The usual working up procedure gave 8.7 g (86.7%) of titlecompound, m.p. 156-159° C., [α]_(D) +48° (c 0.32; CHCl₃).

[0157] The ¹³C NMR spectrum of the title compound is as follows:

[0158] 179.5, 171.1, 170.9, 80.9, 73.5, 64.5, 55.4, 51.7, 50.6, 41.0,40.3, 38.4, 37.8, 37.3, 37.1, 32.6, 27.9, 25.9 (2 C), 23.6, 21.3, 20.9,20.8, 19.9, 18.0, 16.5, 16.3, 16.1,16.0.

Example 8 (pivaloliox)methyl-3β,28-diacetoxy-18-hydroxy-19,20,21,29,30-pentanorlupan-22-oate

[0159] DBU (0.3 g; 2 mmol) and chloromethylpivalate (0.3 g; 2 mmol) wereadded to the solution of3β,28-diacetoxy-18-hydroxy-19,20,21,29,30-pentanorlupan-22-oic acid (1g; 2 mmol) in a mixture of dichloromethane (5 ml) and acetonitrile (2ml). The mixture was vigorously stirred for 3 hours and then dilutedwith an ice-cold water and extracted with chloroform (3×, 10 ml).Collected organic extracts were washed with cold brine, dried andchloroform was evaporated in vacuum. The resulting viscous pale yellowoil (1.5 g) was chromatographed on silicagel, eluting with toluene.After the crystallization from methanol, 0.6 g (48%) of the titlecompound was obtained in the form of colorless needles, m.p. 235-240°C., [α]_(D) +53° (c 0.23; CHCl₃).

[0160] The ¹³C NMR spectrum obtained for the title compound is asfollows:

[0161] 177.4, 172.9, 171.0, 170.6, 80.8, 80.0, 73.5, 64.1, 55.4, 52.1,50.6, 41.0, 40.3, 38.8, 38.5, 37.8, 37.3, 37.1, 32.7, 27.9, 26.8 (3 C),25.9, 25.8, 23.6, 21.3, 20.9, 20.7, 19.8, 18.1, 16.5, 16.3, 16.1, 16.0.

[0162] The following esters were prepared via this general procedure:

[0163](pivaloyloxy)methyl-3β,28-diacetoxy-18-oxo-18,19-seco-19,20,29,30-tetranorlupan-21-oate

[0164] (pivaloyloxy)methyl-3β-hydroxy-30-oxo-lup-20(29)-en-28-oate

[0165] (pivaloyloxy)methyl-3β-acetoxy-21-oxo-lup-18-en-28-oate

[0166] In the same manner, using AcM-Br instead of POM-Cl, followingesters were prepared:

[0167]Acetoxymethyl-3β,28-diacetoxy-18-hydroxy-19,20,21,29,30-pentanorlupan-22-oate

[0168]Acetoxymethyl-3β,28-diacetoxy-18-oxo-18,19-seco-19,20,29,30-tetranorlupan-21-oate

[0169] Acetoxymethyl-3β-hydroxy-30-oxolup-20(29)-en-28-oate

[0170] Acetoxymethyl-3β-acetoxy-21-oxolup-18-en-28-oate

Example 9 3β,28-diacetoxy-18-oxo-19,20,21,29,30-pentanorlup-12-en-22-oicacid

[0171] Bis(tributyltin) oxide (1.9 g; 3.2 mmol) was added to a solutionof (pivaloyloxy)methyl3β,28-diacetoxy-18-oxo-19,20,21,29,30-pentanorlup-12-en-22-oate (1 g;1.6 mmol) and AIBN (20 mg) in ether (50 ml). The mixture was vigorouslystirred until the reaction was completed (TLC was developed withchloroform/ethyl acetate).

[0172] Ether was then evaporated under vacuum and the product waspurified by chromatography on silicagel, eluting with chloroform/ethylacetate. After crystallization from a dichloromethane/ether mixture thetitle compound was obtained in the form of white crystalline solid (0.5g, 62%), m.p. 138-141° C., [α]_(D) +38° (c 0.25; CHCl₃).

[0173] The ¹³C NMR spectrum of the title compound is as follows:

[0174] 16.0, 16.7, 16.9, 18.1, 20.8, 21.3, 23.4, 24.2, 24.4, 24.6, 25.2,27.8, 33.4, 36.8, 37.7, 38.3, 38.7, 43.9, 46.9, 55.4, 58.9, 64.3, 80.5,138.1, 140.6, 170.5, 170.9, 171.6, 196.8.

Example 10 3β,28-diacetoxy-18-oxo-19,20,21,29,30-pentanorlup-22-oylFluoride and Similar Compounds

[0175] Diethylaminosulphur trfluoride (0.5 ml; 3.25 mmol) was addeddropwise to a solution of3β,28-diacetoxy-18-oxo-19,20,21,29,30-pentanorlup-22-oic acid (0.5 g;1.0 mmol) in dry chloroform (5 ml) and the reaction mixture was stirredovernight at room temperature. After the reaction was complete, themixture was slowly poured into cold water (50 ml) and extracted twicewith chloroform. The combined organic fractions were worked up in thegeneral manner and chromatographed on silica gel (20% ethyl acetate inhexane). The residue was crystalized from isopropyl alcohol to give 0.19g (38%) of the title compound as white crystals, m.p. 150-155° C.(decomp.), [α]_(D) +21° (c 0.55; CHCl₃).

[0176] The ¹³C NMR spectrum of the title compound is as follows:

[0177] 211.3 s, 160.2 d (J=374), 170.1 s, 169.5 s, 80.6 s, 65.4 s, 57.5d (J=39), 55.1 s, 50.6 s, 49.9 s, 46.8 s, 40.1 s, 38.5 s, 37.8 s, 36.1s, 33.3 s, 28.4 s, 27.9 s, 26.8 s, 23.5 s, 21.3 s, 21.2 s, 20.6 s, 19.6s, 18.0 s, 16.5 s, 16.4 s, 16.2 s, 15.9 s.

Example 11

[0178] Biological Activity of Betulinines

[0179] 11.1. In Vitro Cytotoxic Activity of Betulinines on Tumor CellLines

[0180] One of the parameters used as the basis for colorimetric assaysis the metabolic activity of viable cells. For example, a microtiterassay which uses the tetrazolium salt MTT is now widely used toquantitate cell proliferation and cytotoxicity [Hajdúch M, Mihál V,Minarík J, Fáber E, {haeck over (S)}afárová M, Weigl E, Antálek P.:Cytotechnology, 1996, 19, 243-245]. For instance, this assay is used indrug screening programs and in chemosensitivity testing. Becausetetrazolium salts are cleaved only by metabolically active cells, theseassays exclusively detect viable cells. In the case of the MTT assay,yellow soluble tetrazolium salt is reduced to a coloured water-insolubleformazan salt. After it is solubilized, the formazan formed can easilyand rapidly be quantified in a conventional ELISA plate reader at 570 nm(maximum absorbancy). The quantity of reduced formazan corresponds tothe number of vital cells in the culture.

[0181] Human T-lymphoblastic leukaemia cell line CEM was used forroutine screening of these compounds. To prove a common mechanism ofaction, selected compounds which showed activity in a screening assaywere tested in a panel of cell lines (Table 2). These lines were fromdifferent species and of different histogenetic origin and they possessvarious alterations in cell cycle regulatory proteins and hormonedependence status (Table 2). The cells were maintained in Nunc/Corning80 cm² plastic tissue culture flasks and cultured in cell culture medium(DMEM with 5 g/l glucose, 2 mM glutamine, 100 U/ml penicillin, 100 μg/mlstreptomycin, 10% foetal calf serum and sodium bicarbonate). Individualcompounds were dissolved in 10% diiethylsulfoxide/saline, pH 8.0.

[0182] The cell suspensions that were prepared and diluted according tothe particular cell type and the expected target cell density(2,500-30,000 cells per well based on cell growth characteristics) wereadded by pipette (80 μl) into 96/well microtiter plates. Inoculates wereallowed a pre-incubation period of 24 hours at 37° C. and 5% CO₂ forstabilisation. Four-fold dilutions of the intended test concentrationwere added at time zero in 20 μl aliquots to the microtiter plate wells.Usually, test compounds were evaluated at six 4-fold dilutions. Inroutine testing, the highest well concentration was 250 μM, but it maydiffer, depending on the agent. All drug concentrations were examined induplicate. Incubations of cells with the test compounds lasted for 72hours at 37° C., in 5% CO₂ atmosphere and 100% humidity. At the end ofthe incubation period, the cells were assayed by using the MTT assay.Ten microliters of the MIT stock solution were pipetted into each welland incubated further for 1-4 hours. After this incubation period,formazan was solubilized by the addition of 100 μl/well of 10% SDS inwater (pH=5.5) followed by further incubation at 37° C. overnight Theoptical density (OD) was measured at 540 nm with the Labsystem iEMSReader MF(UK). The tumour cell survival (TCS) was calculated using thefollowing equitation: TCS=(OD_(drug exposed well)/meanOD_(control wells))×100%. The TCS₅₀ value, the drug concentration lethalto 50% of the tumour cells, was calculated from the obtained doseresponse curves.

[0183] To evaluate the anti-cancer activity of betulinines, theircytotoxic activity against CEM cell line was examined using thescreening assay. Potent compounds were further tested against a panel ofcell lines of different histogenetic and species origin (Table 2). TABLE2 Cytotoxic activity of selected betulinines against a panel ofdifferent (non)malignant cell lines. Compound (TCS₅₀[μM]) Betulinic CellLine Description acid I.3 I.7 I.28 I.44 I.55 I.57 B16 mouse melanoma 362.1 B16F mouse melanoma, metastatic 4.6 4.7 SW620 human colon cancer,250 1.2 metastasis U87MG human glioblastoma 250 5.1 HepG2 humanhepatocellular 3.6 1.7 carcinoma A549 human lung adenocarcinoma 236 1.0MCF-7 human breast cancer, estrogen 194 2.3 dependent, p53+/+, Rb+/+U2OS human osteosarcoma, p53+/−, 250 1.5 Rb+/− Saos2 humanrhabdomyosarcoma, 250 1.8 p53−/−, Rb−/− BT549 human breast cancer, 2502.0 p53mut/mut MDA-MB-238 human breast cancer, estrogen 195 1.4independent, p53mut/mut DU145 human prostate cancer, 241 0.8 androgenindependent, Rb−/− HT-29 human colon cancer 250 1.6 OVCAR-3 humanovarian cancer 164 1.0 Caco-2 human colon cancer 20 3.0 MEL-3 humanmelanoma 2.7 1.3 Lymphocytes human normal lymphocytes 250 13 NIH3T3mouse immortalised fibroblasts 250 7.2 K562 human promyelocytic leukemia250 0.2 K562-CdA human promyelocytic leukemia, 250 0.3 cladrubinresistant K562-GEM human promyelocytic leukemia, 101 0.9 gemcitabinresistant K562-ARA-C human promyelocytic leukemia, 250 0.6 cytarabinresistant K562-FLUD human promyelocytic leukemia, 250 0.4 fludarabinresistant CEM human T-lymphoblastic 250 1.0 5.0 18 11 26 0.2 leukemiaCEM-DNR human T-lymphoblastic 250 0.6 1/C2 leukemia, daunorubicinresistant CEM-DNR human T-lymphoblastic 250 1.1 bulk leukemia,daunorubicin resistant CEM-VCR human T-lymphoblastic 19 3.3 1/F3leukemia, vincristin resistant CEM-VCR human T-lymphoblastic 24 2.9 3/D5leukemia, vincristin resistant CEM-VCR human T-lymphoblastic 69 2.5 bulkleukemia, vincristin resistant

[0184] In contrast to betulinic acid, which is reported to be an agentselective for neuroectodermal derived tumours, there was no significantdifference in sensitivity of betulinines to tumours of differenthistogenetic origin.

[0185] The compounds are effective in submicromolar or low micromolarconcentrations. However, the non-malignant cells, e.g. NIH3T3fibroblasts and normal human lymphocytes, tolerated substantially higherdoses of betulinines than the tumour cells suggesting a favourabletherapeutic index.

[0186] Notably, the effectiveness of betlilines was found to beidentical in cell lines bearing vanous mutations or deletions in cellcycle associated proteins (Table 2). This indicates that thesesubstances should be equally effective in tumours with variousalterations of tumour suppresser genes, namely p53, Rb, etc.

[0187] Furthermore, betulinines were shown to be equally effective indrug resistant cell lines as on their maternal counterparts, therebysuggesting that classical mechanisms of multidrug resistance apparentlydo not apply to these compounds. This particular characteristic shouldbe of significant therapeutic benefit to chemotherapy resistant cancerpatients.

[0188] Finally, the cytotoxic activity of betulinines is independent ofthe hormonal status of cancer cells, so the compounds should be equallyeffective in treatment of hormone dependent and independent cancers.

[0189] 11.2. Betulinines Induce Apoptosis in Tumour Cells.

[0190] To analyse the mechanisms of betulinine-induced cytotoxicity, itis important to distinguish apoptosis from the other major form of celldeath, necrosis. Finally, at the tissue level, apoptosis produces littleor no inflammation, since shrunken portions of the cell are engulfed bythe neighbouring cells, especially macrophages, rather than beingreleased into the extracellular fluid. In contrast, in necrosis,cellular contents are released into the extracellular fluid, and thushave an irritant affect on the nearby cells, causing inflammation.Secondly, at the cellular level, apoptotic cells exhibit shrinkage andblebbing of the cytoplasm, preservation of structure of cellularorganelles including the mitochondria, condensation and margination ofchromatin, fragmentation of nuclei, and formation of apoptotic bodies,thought not all of these are seen in all cell types. Thirdly, at themolecular level, a number of biochemical processes play an importantrole in the induction of apoptosis. However, the majority of them arenot well understood, and they result in activation of proteases andnucleases, which finally destruct key biological macromolecules—proteinsand DNA. For the detection of apoptotic versus necrotic modes of celldeath, the morphology was assessed by scanning electron microscopy.

[0191] A549 cell line was cultured on tissue culture treated glasscoverslips in 6-well culture plates with or without 2 μM concentrationof I.3 or betulinic acid at 37° C. and 5% CO₂ for 12 hours. Followingincubation, the coverslips were washed in Hank's buffered salt solutionand processed as described below.

[0192] Cells were fixed in 2% glutaraldehyde/PBS overnight at 4° C.,dried and covered with gold under vacuum. The surface of the cells wasexamined for typical morphologic markers of apoptosis under a scanningelectron microscope (Tesla, Czech Republic).

[0193] Initial phase contrast microscopy examination indicated thatbetulinines induce typical morphological features of apoptosis in cancercells. This was later confirmed by electron microscopy (FIG. 1). Typicalmorphological criteria of apoptosis were identified in cells treatedwith betulinine I.3: cytoplasmatic blebbing, cellular fragmentation andformation of apoptotic bodies.

[0194] 11.3. In Vivo Activity of Betulinine I.3.

[0195] Animal tumour systems are important models for determining theability of a compound to be adsorbed into the blood stream, to penetrateinto the tumour compartment and to kill fractions of proliferating orresting tumour cells at minimally toxic doses. Although subject tocriticism, in vitro/in vivo models have identified all of thechemotherapeutic agents effective in current clinical practice. It iswell recognised that most compounds are efficacious in lymphomas andleukemias, whereas few have proved to be effective in solid tumours,illustrating that there are major differences between animal models andclinical situations. Solid tumours of different histogenetic origin areimplanted subcutaneously or intramuscularly into animals which are thentreated with a single agent. Inhibition of solid tumour growth is thus aparameter related to the activity of the drug. Conversely, in patientsprimary tumours are controlled by surgery and radiation, together withpolychemotherapy. Usually, the acceptable criterion for activity is >50%tumour regression. However, drugs active in several different screeningsystems are more likely to be effective in humans; for instance,doxorubicin, cyclophosphamide and cisplatin have a broad spectrum ofactivity in animal tumour models. Moreover, with regard to toxicity andtotal drug exposure, a correlation has been found between nice and manfor most active antitumor drugs.

[0196] Betulinic acid was reported to exhibit prominent activity inneuroectodermal tumours, e.g. melanoma, primitive neuroectodermaltumours (PNET) and neuroblastoma. The latter is the most lethal solidtumour of childhood as it is considered to be one of the most drugresistant tumours. In the light of this knowledge, the activity of I.3in xenotransplanted human neuroblastoma was of particular interest,although this compound demonstrated broad anti-cancer activity under invitro conditions.

[0197] Human neuroblastoma SK-N-AS cell line was obtained from ATTC. Theline was cultured in Dulbeco's modified essential medium with 4.5 gdextrose/l, 10% of foetal calf serum, 2 mM glutamine, 100 U/mlpenicillin and 100 mg/ml streptomycin. For transplantation purposes,only the cells in log phase were used.

[0198] CD-1/Ctrl-1 nu/nu nude female mice, 8 weeks old, 18-22 g weightwere used in this study (IffaCredo, France). Animals were inoculatedwith 5.10⁶ tumour cells subcutaneously into right inquinal region.Following transplantation, tumor growth was measured in both thecontrol/vehicle and the treated groups using calipers. Tumor volume (TV)was calculated as follows: TV=(a²×b)/2, where a is width and b is lengthThe statistical significance was evaluated using a non-parametrict-test.

[0199] Cisplatin (Platidiam 10 inj, sicc., Lachema, Czech Republic) wasused as a control drug in this experiment. It was diluted in apyrogenicwater and applied to animals in a final volume of 0.18 ml subcutaneouslyat day 1.

[0200] I.3 was synthesised as described above. It was suspended at 10mg/ml in 5% dextrose and pH was adjusted to 8.0 with sodium hydroxide.

[0201] vehicle: 5% dextrose with pH adjusted to 8.0 with sodiumhydroxide.

[0202] Animals were treated with drugs/vehicle 24 hours aftertransplantation The following groups (with 10 animals per group) werecreated:

[0203] control—untreated animals

[0204] vehicle—0.2 ml of vehicle applied to animals at 3×D1,2,5,6,9,10intraperitoneally

[0205] cisplatin 4 mg/kg—0.18 ml of cisplatin solution applied toanimals subcutaneously at 1×D1

[0206] I.3 1 mg/mouse s.c.—0.1 ml of I.3 suspension applied to animalsat 3×D1,2,5,6,9,10 subcutaneously

[0207] I.3 2 mg/mouse s.c.—0.2 ml of I.3 suspension applied to animalsat 3×D1,2,5,6,9,10 subcutaneously

[0208] I.3 1 mg/mouse i.p.—0.1 ml of I.3 suspension applied to animalsat 3×D1,2,5,6,9,10 intraperitoneally

[0209] I.3 2 mg/mouse i.p.—0.2 ml of I.3 suspension applied to animalsat 3×D1,2,5,6,9,10 intraperitoneally

[0210] The results of the study are summarised in FIG. 2. The anticanceractivity of I.3 is clearly demonstrated, since there is no tumor growthin I.3 treated animals. It was statistically significant in all I.3applied groups starting from week 2 of the whole experiment. The effectwas independent of the application route (intraperitoneal orsubcutaneous) and of the dose in this application.

[0211] In contrast to I.3, cisplatin was ineffective in the managementof SK-N-AS neuroblastoma and showed no significant activity.

[0212] The anticancer activity of I.3 as a typical representative ofthis generation of betulinines was demonstrated under in vivoconditions. This novel compound seems to be highly active against humanneuroblastoma under in vivo conditions. Together with its broadanticancer activity and novel, previously unidentified mechanism ofaction, the compounds of the present invention are believed to be oftherapeutic potential for cancer patients in the future.

[0213] 11.4. Betulinine I.3 Induces Rapid Dephosphorylation of RbProtein and Apotosis Related Caspase Activation.

[0214] As discussed earlier, the G1/S transition is tightly regulated byphosphorylation of retinoblastoma protein (Rb).

[0215] Since Rb protein contains multiple phosphorylation sites forCDKs, its phosphorylated form has molecular weight about 110 kDa, whilethe molecular weight of hypophosphorylated protein is only 105 kDa Thissmall difference in molecular weight is enough to separate both forms byconventional SDS-PAGE electrophoresis.

[0216] CEM cells were cultured in Dulbeco's modified essential mediumwith 4.5 g dextrose/l, 10% of foetal calf serum, 2 mM glutamine, 100U/ml penicillin and 100 μg/ml streptomycin with/without below indicatedconcentrations of betulkic acid (BA) or I.3. At selected time points,cells were harvested, washed in ice cold Hank's balanced salt solutionand solubilized on ice using the SDS-PAGE sample buffer containingprotease and phosphatase inhibitors (10 μg/ml of leupeptin, 10 μg/ml ofaprotinin, 10 μg/ml of soybean trypsin inhibitor, 100 μmol of benzamide,1 mM of sodium vanadate, 1 mM of NaF, 1 mM of phenylphosphate) andboiled immediately.

[0217] Total cellular proteins (100 μg/well) were separated on SDS-PAGEelectrophoresis, blotted on polyvinyldifluoride membranes and total Rbprotein, including proteolytic fragment(s) were detected using a pRbmonoclonal antibody (Oncogene, Germany, Rb(AS5), Cat# OP66 Rev 2,Sep.1996 EB, Clone LM95.1) and visualized by chemilumniuscence (ECL-WestegBlotting System, Amersham). Details of the Western blot technique aredescribed in Ausubel, F. M., Brent, R., Kingston, P E., Moore, D. D.,Seidman, J. G., Smith, J. A., Struhl, K (Eds): Short Protocols-inMolecular Biology, 2nd edition, John Wiley & Sons, New York, Chichester,Brisbane, Toronto, Singapore, 1992, page 10-33-10-35.

[0218] The results of this study indicate that in CEM lymphoblasticleukaemia cells, Rb protein is rapidly dephosphorylated followingtreatment with I.3, but not betulinic acid (FIG. 3). This isdemonstrated by a shift of Rb protein mass from the hyperphosphorylatedform with a molecular mass of about 110 kDa to the hypophosphorylatedform (105 kDa). The effect of I.3 is time and concentration dependent;at 20 μM concentration, the hypocoincident form of Rb appears as earlyas 15 minutes after the treatment, while at 7 μM the samny effectappears after 2 hours. Interestingly, cleavage of the Rb protein afterhypophosphorylation was accompanied with the disappearance of a 105 kDaRb and the appearance of an immunoreactive fragment of Rb protein withmolecular weight about. 42 kDa. According to the literature, proteolyticdecomposition of Rb is a typical hallmark of apoptosis due to activationof cellular caspases.

[0219] The results of this study clearly indicate that the betulininesof the present invention, but not betilinic acid itself are capable ofinducing the rapid dephosphorylation of key cell cycle regulatoryprotein Rb. This is followed by the induction of apoptosis, which hasbeen reported to activate cellular caspases. Activated caspases have thecapacity to cleave target proteins, including Rb. This is illustrated bythe time and concentration dependent appearance of an immunoreactivefragment of Rb protein with molecular weight 42 kDa in I.3 treatedcells.

[0220] 11.5. Betulinine I.3 Induces G1 Block and Apoptosis in TumourCells.

[0221] Hypophosphorylation of Rb protein is accompanied with cell cycleblock on the G1/S transition. To investigate whether incubation oftumour cells with betulinines will result in cell cycle block and/orapoptosis, a flow-cytometry study was performed with measurement oftotal DNA content in CEM cells treated with/without I.3. Taxol(paclitaxel) was used as a positive control, since this drug is known toresult in the cumulation of cells in the G2 phase.

[0222] Briefly, CEM cells were cultured in Dulbeco's modified essentialmedium with 4.5 g dextrose/l, 10% of foetal calf serum, 2 mM glutamine,100 U/ml penicillin and 100 μg/ml streptomycin with/without theindicated concentrations of paclitaxel or I.3 (FIG. 4A). At selectedtime points, cells were harvested, washed in ice cold Hank's balancedsalt solution (HBSS) and fixed in 70% ethanol at −20° C. overnight. Thenext day, ethanol was removed by centrigation, cells were washed twicein HBSS and a cellular pellet (10⁶ cells) was reconstituted in stainingsolution (propidium iodide 60 μg/ml, DNA-se free RNA-se 175 U/ml inHBSS) for 15 minutes at 37° C. The DNA content of individual cells wasanalyzed on flow cytometer Excalibur (Becton and Dickinson) atexcitation wavelength of 488 nm.

[0223] As indicated in FIG. 4A,B I.3 induces cumulation of cells inG0/G1 phases of the cell cycle, which is accompanied by rapid apoptosis(appearance of sub-G1 cells). The effect is concentration and timedependent. Induction of G1 block and apoptosis is consistent withdephosphorylation of Rb protein

[0224] 11.6. Betulinine I.3 Induces Rapid Histone Acetylation.

[0225] Cellular cytotoxicity studies (data not shown) demonstrated thatthe cytotoxic activity of I.3 strongly correlates with actinomycin D(p<0.00001). Since actinomycin D is a well known inhibitor of RNApolymerases, these results suggest that I.3 is targeting transcriptionalcomplexes.

[0226] As discussed earlier, the G1/S transition is tightly regulated byphosphorylation of retinoblastoma protein (Rb). Hypophosphorylated Rbsilences specific genes that are active in the S phase of the cell cycleand which are regulated by E2F transcription factors. Rb binds to theactive domain of E2F and then actively repress the promoter by amechanism that is poorly understood. Recent studies show that Rbassociates with a histone deacetylase (HDAC 1 and 2) through the Rbpocket domain. Rb recruits histone deacetylase to E2F and thus Rbcooperates with HDAC to repress the E2F-regulated promotor of the gene.Inhibition of histone deacetylase activity by specific inhibitortrichostatin A (TSA) inhibits Rb-mediated repression of a E2Ftranscriptional activity.

[0227] In order to provide the evidence of interference of betulinineswith transcription, the ability of I.3 to modify histone acetylation wasinvestigated.

[0228] CEM cells were cultured in Dulbeco's modified essential mediumwith 4.5 g dextrose/l, 10% of foetal calf serum, 2 mM glutamine, 100U/ml penicillin and 100 μg/ml streptomycin with/without the indicatedconcentrations of HDAC inhibitor TSA or I.3. At selected time points,cells were harvested, washed in ice cold Hank's balanced salt solutionand solubilized on ice using SDS-PAGE sample buffer containing proteaseand phosphatase inhibitors (10 Wg/ml of leupeptin, 10 μg/ml ofaprotinin, 10 μg/ml of soybean trypsin inhibitor, 100 pmol of benzamide,1 mM of sodium vanadate, 1 mM of NaF, 1 mM of phenylphosphate) andboiled immediately.

[0229] Total cellular proteins (100 μg/well) were separated on SDS-PAGEelectrophoresis, blotted on polyvinyldifluoride membranes and acetylatedhistone was detected using a anti-acetyl(Lys9)-histone H3 rabbitpolyclonal antibody (Cell Signalling Technology, Beverly, Mass.; Cat#9671L) and visualized by chemilumniuscence (ECL-Western Blotting System,Amersham). Details of the Western blot technique are described inAusubel, F. M., Brent, R, Kingston, R. E., Moore, D. D., Seidman, J. G.,Smith, J. A., Struhl, K. (Eds): Short Protocols in Molecular. Biology,2nd edition, John Wiley & Sons, New York, Chichester, Brisbane, Toronto,Singapore, 1992, page 10-33-10-35.

[0230] The results of this study indicate that in CEM lymphoblasticleukaemia cells, H3 histone is rapidly acetylated in Lys9 positionfollowing the treatment of both TSA and betulinine I.3 (FIG. 5). Theeffect of I.3 is time and concentration dependent; at 20 μM (10×IC₅₀)acetylated histone appears as soon as 30 minutes after incubation.Interestingly, CEM cells cultured with 2 μM of I.3 (IC₅₀) show histoneacetylation in time points in between 45-120 minutes, which isconsistent with degradation half-life of I.3 under in vitro conditions(data not shown). On the other hand, however, TSA at 0.24 μM (IC₅₀)showed no significant ability to increase hustone acetylation,suggesting that mechanism(s) of cytotoxic activity of TSA may be morecomplex tat we originally realised.

[0231] The results of this study clearly indicate that the betulininesof the present invention, are capable of inducing the rapid histoneacetylation. It is generally accepted that N termini of core histonesare central to the processes that modulate nucleosome structure.Hyperacetylation of the histones reduces their ability to constrain thepath of DNA within chromatin, resulting in allosteric changes innucleosomal conformation, destabilization of internucleosomal contacts,and an increase in the accessability of nucleosomal DNA to transcriptionfactors. The amount of histone acetylation is determined by anequilibrium between histone acetyltransferases (HAT) and histonedeacetylases (HDAC). The balance plays an important role in theregulation of gene transcription as well as the genesis or suppressionof cancer. Unfortunately, the question of whether betulinines inhibitHDAC directly or indirectly remains open. As discussed earlier,betulinines induce rapid dephosphorylation of Rb protein.Hypophosphorylated Rb was shown to bind both HDAC and E2F-DP1 complex.It has been recently reported that E2F-1, -2, and -3, but not E2F-4, -5,and -6, associate with and are acetylated by p300 and cAMP-responseelement-binding protein acetyltransferases. Acetylation occurs at threeconserved lysine residues located at the N-terminal boundary of theirDNA binding domains. Acetylation of E2F-1 in vitro and in vivo markedlyincreases its binding affinity for a consensus E2F DNA-binding site,which is paralleled by enhanced transactivation of an E2F-responsivepromoter. Acetylation of E2F-1 was reversed by HDAC1, indicating thatreversible acetylation is a mechanism for regulation also of non-histoneproteins. Thus, inhibition of HDAC by synthetic compounds should resultin both histone and transcription factor acetylation resulting intranscriptional activation of responsive genes. Interestingly, a numberof oncosupressors and virus promotor driven genes are transcritionallyrepressed in cancer/nectvirus infected cells due to constitutivedeacetylation of specific promoters. Inhibition of HDAC results inre-activation of onco-suppressors and genes under virus promotor. Thoseskilled in the art will recognise that betulinines could be used notonly in treatment of cancer, but also in the treatment of diseases or intherapeutic approaches, where transcriptional repression of specificgene(s) is undesired, for instance transcriptional silencing of vectorsused in gene therapy.

[0232] Notably, since these potential drugs do not exhibit any CDKinhibitory activity (data not shown), we expect that they possess a new,previously unreported mechanism of action.

[0233] Those skilled in the art will recognise, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the claims.

1. A method of treating a proliferative disease comprising administering a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof

wherein: X¹ is C═O, C═NOR^(1a), CHOR^(1a), CHOCOR^(1a), CHOCOY-Hal, CHOC(O)OR⁹, CHOC(O)OR^(1a), CHOC(O)OR¹⁰, and Hal is Br, Cl, I, F; X³ is C═O, CHOR^(1b), or CHOCOR^(1b); R¹⁻⁵ are each independently H or lower alkyl; R⁶ is H or absent if “a” is a double bond; R⁷ is H, COOR^(1c), YOCOR^(1c), COOYOCOR^(1e), YCOOR^(1e); R⁸ is H, COOR^(1d), YCOOR^(1d), YCOOR^(1d), YCOHal, COOYOCOR^(1d), CH₂OR^(1d), CH₂COCOR^(1d), COCOCOR^(1d), or R⁷ and R⁸ together are ═CH₂; R⁹ is an OH-substituted alkyl group, an ether group or a cyclic ether; R¹⁰ is lower alkyl substituted Hal; “a” is a double bond or single bond; and wherein Y═(CH₂)_(n) n=0 to 5; R^(1a-1d) are the same or different groups of R¹. 2-21. (Cancelled).
 22. The method of claim 1, wherein: X¹ is CHOR^(1a), CHOCOR^(1a) or CHOCOY-Hal; and R⁸ is H, COOR^(1d), YCOOR^(1d), COOYOCOR^(1d), CH₂OR^(1d), CH₂COCOR^(1d) or COCOCOR^(1d).
 23. The method of claim 1, wherein: R²⁻⁵ are all methyl and R¹ is as defined below for the relevant group R^(1a-1d); X¹ is —CHOCOCH₂Cl; or —CHOR^(1a)OR CHOCOR^(1a), wherein R^(1a) is H and methyl respectively; X³ is C═O, CHOH or CHOAc; R⁷ is H, COOH, COOMe, CH₂OAc, COOYOCOR^(1e) or YCOOR^(1e) where Y is CH₂ and R^(1e) is C₁₋₄ alkyl; R⁸ is —COOR^(1d), wherein R^(1d) is H or methyl; —YCOOR^(1d), wherein Y is CH₂ and R^(1d) is H, methyl or ethyl —COOYOCOR^(1d), wherein Y is CH₂ and R^(1d) is C₁₋₄ alkyl; —CH₂OR^(1d), wherein R^(1d) is C₁₋₄ alkyl; CH₂COCOR^(1d) or COCOCOR^(1d), wherein R^(1d) is C₁₋₄ alkyl.
 24. The method of claim 22, wherein: R²⁻⁵ are all methyl and R¹ is as defined below for the relevant group R^(1a-1d); X¹ is —CHOCOCH₂Cl; or —CHOR^(1a)OR CHOCOR^(1a), wherein R^(1a) is H and methyl respectively; X³ is C═O, CHOH or CHOAc; R⁷ is H, COOH, COOMe, CH₂OAc, COOYOCOR^(1e) or YCOOR^(1e) where Y is CH₂ and R^(1e) is C₁₋₄ alkyl; R⁸ is —COOR^(1d), wherein R^(1d) is H or methyl; —YCOOR^(1d), wherein Y is CH₂ and R^(1d) is H, methyl or ethyl —COOYOCOR^(1d), wherein Y is CH₂ and R^(1d) is C₁₋₄ alkyl; —CH₂OR^(1d), wherein R^(1d) is C₁₋₄ alkyl; CH₂COCOR^(1d) or COCOCOR^(1d), wherein R^(1d) is C₁₋₄ alkyl.
 25. The method of claim 23, wherein R⁸ is —COOYOCOR^(1d), wherein Y is CH₂ and R^(1d) is methyl or butyl; —CH₂OR^(1d), wherein R^(1d) is methyl or ethyl; —CH₂COCOR^(1d) or COCOCOR^(1d), wherein R^(1d) is propyl; and R⁷ is COOYOCOR^(1e) or YCOOR^(1e) where Y is CH₂ and R^(1e) is methyl or butyl.
 26. The method of claim 24, wherein R⁸ is —COOYOCOR^(1d), wherein Y is CH₂ and R^(1d) is methyl or butyl; —CH₂OR^(1d), wherein R^(1d) is methyl or ethyl; —CH₂COCOR^(1d) or COCOCOR^(1d), wherein R^(1d) is propyl; and R⁷ is COOYOCOR^(1e) or YCOOR^(1e) where Y is CH₂ and R^(1e) is methyl or butyl.
 27. The method of claim 1 wherein “a” is a single bond and R⁶ is H.
 28. The method of claim 22 wherein “a” is a single bond and R⁶ is H.
 29. The method of claim 23 wherein “a” is a single bond and R⁶ is H.
 30. The method of claim 24 wherein “a” is a single bond and R⁶ is H.
 31. The method of claim 25 wherein “a” is a single bond and R⁶ is H.
 32. The method of claim 26 wherein “a” is a single bond and R⁶ is H.
 33. A method of treating a proliferative disease comprising administering a compound of formula I, or a pharmaceutically acceptable salt thereof,

wherein the compound is selected from the group consisting of: No. X¹ R¹ X³ a R²⁻⁵ R⁶ R⁷ R⁸ 1.1 CHOAc CH₃ C═O single Me H CH₂OAc CH₂COCOPr¹ 1.2 CHOAc CH₃ C═O single Me H CH₂OAc CH₂COCOPr¹ 1.3 CHOAc CH₃ C═O single Me H CH₂OAc COOH 1.4 CHOAc CH₃ C═O single Me H CH₂OAc COOMe 1.5 CHOAc CH₃ C═O single Me H H H 1.6 CHOAc CH₃ C═O single Me H H CH₂COOH 1.7 CHOAc CH₃ C═O single Me H H CH₂OEt 1.8 CHOAc CH₃ CHOH single Me H CH₂OAc COOH 1.9 CHOAc CH₃ C═O double Me absent CH₂OAc COOMe 1.10 CHOAc CH₃ CHOH single Me H R⁷ and R⁸ together: ═CH₂ 1.11 CHOAc CH₃ CHOAc single Me H CH₂OAc COOMe 1.12 CHOAc CH₃ C═O single Me H COOMe CH₂COCOPr¹ 1.13 CHOAc CH₃ C═O single Me H COOMe COOH 1.14 CHOAc CH₃ C═O single Me H COOMe CH₂COOH 1.15 CHOAc CH₃ CHOH single Me H CH₂OAc COOCH₂OCOBu^(t) 1.16 CHOAc CH₃ CHOH single Me H CH₂OAc COOMe 1.17 CHOAc CH₃ C═O single Me H CH₂OAc CH₂COOH 1.18 CHOAc CH₃ C═O single Me H CH₂OAc COOCH₂OCOBu^(t) 1.19 CHOAc CH₃ C═O single Me H CH₂OAc COOCH₂OCOMe 1.20 CHOAc CH₃ C═O double Me absent CH₂OAc COOCH₂OCOBu^(t) 1.21 CHOAc CH₃ C═O double Me absent CH₂OAc COOCH₂OCOMe 1.22 CHOAc CH₃ C═O double Me absent CH₂OAc COOH 1.24 CHOAc CH₃ C═O single Me H R⁷ and R⁸ together ═ CH₂ 1.26 CHOH H C═O single Me H CH₂OAc COOH 1.27 CHOH H C═O double Me absent CH₂OAc COOMe 1.28 CHOAc CH₃ C═O single Me H COOCH₂OCOMe CH₂COCOPr¹ 1.29 CHOAc CH₃ C═O single Me H COOCH₂OCOBut CH₂COCOPr¹ 1.30 CHOAc CH₃ C═O single Me H from R⁷ and R⁸: CH₂OC(O)CH₂ 1.31 CHOAc CH₃ CHOAc single Me H CH₂OAc COOH 1.32 CHOAc CH₃ CHOAc single Me H CH₂OAc COOCH₂OCOBu^(t) 1.33 CHOAc CH₃ CHOH single Me H CH₂OAc COOMe 1.34 CHOAc CH₃ CHOAc single Me H CH₂OAc COOMe 1.35 CHOAc CH₃ CHOH single Me H CH₂Oac COOCH₂OCOBu^(t) 1.36 CHOAc CH₃ CHOAc single Me H CH₂Oac COOCH₂OCOBu^(t) 1.37 CHOAc CH₃ C═O single Me H CH₂Oac COOMe 1.38 CHOAc CH₃ C═O single Me H H CH₂COOMe 1.39 CHOAc CH₃ C═O single Me H COOMe CH₂COOMe 1.40 CHOAc CH₃ C═O single Me H COOCH₂OCOBu^(t) CH₂COOMe 1.41 CHOAc CH₃ C═O single Me H COOCH₂OCOMe CH₂COOMe 1.42 CHOAc CH₃ CHOH single Me H R⁷ and R⁸ together: ═CH₂ 1.43 CHOAc CH₃ CHOH single Me H H H 1.44 CHOCOCH₂Cl CH₃ C═O single Me H CH₂OAc COOH 1.45 CHOAc CH₃ CHOH single Me H CH₂OAc COOH 1.46 CHOAc CH₃ CHOAc single Me H CH₂OAc COOH 1.47 CHOAc CH₃ CHOAc single Me H R⁷ and R⁸ together: ═CH₂ 1.48 CHOAc CH₃ CHOAc single Me H H H 1.49 CHOH CH₃ C═O single Me H H CH₂COOH 1.50 CHOH CH₃ C═O single Me H H CH₂COOMe 1.51 CHOH CH₃ CHOH single Me H H CH₂COOMe 1.52 CHOAc CH₃ CHOH single Me H H CH₂COOMe 1.53 CHOH CH₃ CHOH single Me H CH₂COOMe H 1.54 CHOAc CH₃ CHOAc single Me H CH₂COOMe H

1.57 CHOH CH₃ C═O single Me H CH₂OAc COOH 1.58 CHOAc CH₃ C═O single Me H CH₂OAc COF


34. A method of treating a proliferative disease comprising administering a therapeutically effective amount of a compound selected from the group consisting of:


35. A method of preparing a pharmaceutical composition comprising admixing a compound of formula I and a pharmaceutical carrier, excipient or diluent.
 36. A compound of the formula Ia, or a pharmaceutically acceptable salt thereof;

wherein: X¹ is C═O, C═NOR^(1a), CHOR^(1a), CHOCOR^(1a), CHOCOY-Hal, CHOC(O)OR⁹, CHOC(O)OR^(1a), CHOC(O)OR¹⁰, and Hal is Br, Cl, I, F; X³ is C═O, CHOR^(1b), or CHOCOR^(1b); R¹⁻⁵ are each independently H or lower alkyl; R⁶ is H or absent if “a” is a double bond; R⁷is H, COOR^(1c), YOCOR^(1c), COOYOCOR^(1c), YCOOR^(1c); R⁸ is H, COOR^(1d), YCOOR^(1d), YCOOR¹⁰, YCOHal, COOYOCOR^(1d), CH₂OR^(1d), CH₂COCOR^(1d), COCOCOR^(1d), or R⁷ and R⁸ together are ═CH₂; R⁹ is an OH-substituted alkyl group, an ether group or a cyclic ether; R¹⁰ is lower alkyl substituted Hal; “a” is a double bond or single bond; and wherein Y═(CH₂)_(n) n=0 to 5; R^(1a-1d) are the same or different groups of R¹ with the proviso that (i) when X¹ is CHOAc, X³ is C═O, “a” is a single bond, R¹⁻⁵ are Me, R⁶ is H; when R⁷ is CH₂OAc, R⁸is other than COOH, CH₂COCOPr^(i), COCOCOPr^(i), CH₂COOH or CH₂CH₂COPr^(i); when R⁷ is CO₂Me, R⁸ is other than CH₂CH₂COCH(CH₃)₂; when R⁷ is H, R⁸ is other than H, CH₂COOMe, CH₂COOH or CH₂CH₂COPr^(i); and (ii) when X¹ is CHOH, X³ is C═O, “a” is a single bond, R¹⁻⁵ are Me, and R⁶ is H, when R⁷is H, R⁸is other than H, CH₂COOH, CH₂CH₂COCH(CH₃)₂ or CH₂COOMe; when R⁷is CH₂OAc, R⁸ is other than CH₂COOH; or a pharmaceutically acceptable salt thereof.
 37. The compound of claim 36, wherein: X¹ is CHOR^(1a), CHOCOR^(1a), CHOCOY-Hal; and R⁵ is H, COOR^(1d), YCOOR^(1d), COOYOCOR^(1d), CH2OR^(1d), CH₂COCOR^(1d) or COCOCOR^(1d).
 38. The compound of claim 36, wherein: R²⁻⁵ are all methyl and R¹ is as defined below for the relevant group R^(1a-1d); X¹ is —CHOCOCH₂Cl; or ‘CHOR^(1a) or CHOCOR^(1a), wherein R^(1a) a is H and methyl respectively; X³ is C═O, CHOH or CHOAc; R⁷ is H, COOH, COOMe, CH₂OAc, COOYOCOR^(1e), YCOOR^(1e) where Y is CH₂ and R^(1e) is C¹⁻⁴ alkyl; R⁸ is —COOR^(1d), R^(1d) is H or methyl; —YCOOR^(1d), wherein Y is CH₂ and R^(1d) is H, methyl or ethyl; —COOYOCOR^(1d), wherein Y is CH₂ and R^(1d) is C₁₋₄ alkyl; —CH₂OR^(1d), wherein R^(1d) is C₁₋₄ alkyl; —CH₂COCOR^(1d) or COCOCOR^(1d), wherein R^(1d) is C₁₋₄ alkyl.
 39. The method of claim 37, wherein: R²⁻⁵ are all methyl and R¹ is as defined below for the relevant group R^(1a-1d); X¹ is —CHOCOCH₂Cl; or —CHOR^(1a) or CHOCOR^(1a), wherein R^(1a) a is H and methyl respectively; X³ is C═O, CHOH or CHOAc; R⁷ is H, COOH, COOMe, CH₂OAc, COOYOCOR^(1e), YCOOR^(1e) where Y is CH₂ and R^(1e) is C¹⁻⁴ alkyl; R⁸ is —COOR^(1d), R^(1d) is H or methyl; —YCOOR^(1d), wherein Y is CH₂ and R^(1d) is H, methyl or ethyl; —COOYOCOR^(1d), wherein Y is CH₂ and R^(1d) is C₁₋₄ alkyl; —CH₂OR1d, wherein R^(1d) is C₁₋₄ alkyl; —CH₂COCOR^(1d) or COCOCOR^(1d), wherein R^(1d) is C₁₋₄ alkyl.
 40. The compound of claim 36 wherein: R⁸ is —COOYOCOR^(1d), wherein Y is CH₂ and R^(1d) is methyl or butyl; —CH₂OR^(1d), wherein R^(1d) is methyl or ethyl; —CH₂COCOR^(1d) or COCOCOR^(1d), wherein R^(1d) is propyl; and R⁷ is COOYOCOR^(1e), YCOOR^(1e), where Y is CH₂ and R^(1e) is methyl or butyl.
 41. The compound of claim 37 wherein: R⁸ is —COOYOCOR^(1d), wherein Y is CH₂ and R^(1d) is methyl or butyl; —CH₂OR^(1d), wherein R^(1d) is methyl or ethyl; —CH₂COCOR^(1d) or COCOCOR^(1d), wherein R^(1d) is propyl; and R⁷ is COOYOCOR^(1e), YCOOR^(1e), where Y is CH₂ and R^(1e) is methyl or butyl.
 42. The compound of claim 38 wherein: R⁸ is —COOYOCOR^(1d), wherein Y is CH₂ and R^(1d) is methyl or butyl; —CH₂OR^(1d), wherein R^(1d) is methyl or ethyl; —CH₂COCOR^(1d) or COCOCOR^(1d), wherein R^(1d) is propyl; and R⁷ is COOYOCOR^(1e), YCOOR^(1e), where Y is CH₂ and R^(1e) is methyl or butyl.
 43. The compound of claim 39 wherein: R⁸ is —COOYOCOR^(1d), wherein Y is CH₂ and R^(1d) is methyl or butyl; —CH₂OR^(1d), wherein R^(1d) is methyl or ethyl; —CH₂COCOR^(1d) or COCOCOR^(1d), wherein R^(1d) is propyl; and R⁷ is COOYOCOR^(1e), YCOOR^(1e), where Y is CH₂ and R^(1e) is methyl or butyl.
 44. The compound of claim 36 wherein “a” is a single bond and R⁶ is H.
 45. The compound of claim 37 wherein “a” is a single bond and R⁶ is H.
 46. The compound of claim 38 wherein “a” is a single bond and R⁶ is H.
 47. The compound of claim 39 wherein “a” is a single bond and R⁶ is H.
 48. The compound of claim 40 wherein “a” is a single bond and R⁶ is H.
 49. A compound of formula Ia, or a pharmaceutically acceptable salt thereof

wherein the compound is selected from the group consisting of: No. X¹ R¹ X³ a R²⁻⁵ R⁶ R⁷ R⁸ 1.4 CHOAc CH₃ C═O single Me H CH₂OAc COOMe 1.7 CHOAc CH₃ C═O single Me H H CH₂OEt 1.8 CHOAc CH₃ CHOH single Me H CH₂OAc COOH 1.9 CHOAc CH₃ C═O double Me absent CH₂OAc COOMe 1.10 CHOAc CH₃ CHOH single Me H R⁷ and R⁸ together: ═CH₂ 1.11 CHOAc CH₃ CHOAc single Me H CH₂OAc COOMe 1.12 CHOAc CH₃ C═O single Me H COOMe CH₂COCOPr¹ 1.13 CHOAc CH₃ C═O single Me H COOMe COOH 1.14 CHOAc CH₃ C═O single Me H COOMe CH₂COOH 1.15 CHOAc CH₃ CHOH single Me H CH₂OAc COOCH₂OCOBu^(t) 1.16 CHOAc CH₃ CHOH single Me H CH₂OAc COOMe 1.18 CHOAc CH₃ C═O single Me H CH₂OAc COOCH₂OCOBu^(t) 1.19 CHOAc CH₃ C═O single Me H CH₂OAc COOCH₂OCOMe 1.20 CHOAc CH₃ C═O double Me absent CH₂OAc COOCH₂OCOBu^(t) 1.21 CHOAc CH₃ C═O double Me absent CH₂OAc COOCH₂OCOMe 1.22 CHOAc CH₃ C═O double Me absent CH₂OAc COOH 1.24 CHOAc CH₃ C═O single Me H R⁷ and R⁸ together: ═CH₂ 1.26 CHOH H C═O single Me H CH₂OAc COOH 1.27 CHOH H C═O single Me H CH₂OAc COOMe 1.28 CHOAc CH₃ C═O double Me absent COOCH₂OCOMe CH₂COCOPr¹ 1.29 CHOAc CH₃ C═O single Me H COOCH₂OCOBut CH₂COCOPr¹ 1.30 CHOAc CH₃ C═O single Me H from R⁷ and R⁸: ═CH₂OC(O)CH₂ 1.31 CHOAc CH₃ CHOAc single Me H CH₂OAc COOH 1.32 CHOAc CH₃ CHOAc single Me H CH₂OAc COOCH₂OCOBu^(t) 1.33 CHOAc CH₃ CHOH single Me H CH₂OAc COOMe 1.34 CHOAc CH₃ CHOAc single Me H CH₂OAc COOMe 1.35 CHOAc CH₃ CHOH single Me H CH₂OAc COOCH₂OCOBu^(t) 1.36 CHOAc CH₃ CHOAc single Me H CH₂OAc COOCH₂OCOBu^(t) 1.37 CHOAc CH₃ C═O single Me H CH₂OAc COOMe 1.39 CHOAc CH₃ C═O single Me H COOMe CH₂COOMe 1.40 CHOAc CH₃ C═O single Me H COOCH₂OCOBu^(t) CH₂COOMe 1.41 CHOAc CH₃ C═O single Me H COOCH₂OCOMe CH₂COOMe 1.42 CHOAc CH₃ CHOH single Me H R⁷ and R⁸ together: ═CH₂ 1.43 CHOAc CH₃ CHOH single Me H H H 1.44 CHOC CH₃ C═O single Me H CH₂OAc COOH OCH₂Cl 1.45 CHOAc CH₃ CHOH single Me H CH₂OAc COOH 1.46 CHOAc CH₃ CHOAc single Me H CH₂OAc COOH 1.47 CHOAc CH₃ CHOAc single Me H R⁷ and R⁸ together: ═CH₂ 1.48 CHOAc CH₃ CHOAc single Me H H H 1.51 CHOH CH₃ CHOH single Me H H CH₂COOMe 1.52 CHOAc CH₃ CHOH single Me H H CH₂COOMe 1.53 CHOH CH₃ CHOH single Me H CH₂COOMe H 1.54 CHOAc CH₃ CHOAc single Me H CH₂COOMe H 1.55 CHOAc CH₃ ← single Me H H ← 1.56 CHOH CH₃ ← single Me H H ← 1.57 CHOH CH₃ C═O single Me H CH₂OAc COOH 1.58 CHOAc CH₃ C═O single Me H CH₂OAc COF


50. A method of preparing a compound of formula I, or a pharmaceutically acceptable salt thereof,

wherein “a” is a double bond, the method comprising:

(i) oxidizing a compound of formula 1b to a compound of formula 1c; (ii) reducing said compound of formula 1c to form a compound of formula 1d; and optionally (iii) converting said formula of id to a compound of formula I as defined above.
 51. The method of claim 50, wherein the compound of formula 1b is oxidized to ic by treating sequentially with selenium dioxide, peroxyacetic acid and ruthenium tetroxide.
 52. The method of claim 50, wherein the reduction of 1c to 1d is a stereoselective reduction.
 53. The method of claim 51, wherein the reduction of 1c to 1d is a stereoselective reduction.
 54. The method of claim 50, wherein the compound of formula ic is reduced to 1d by treating with sodium borohydride in the presence of cerium (III) salt.
 55. The method of claim 51, wherein the compound of formula 1c is reduced to Id by treating with sodium borohydride in the presence of cerium (III) salt.
 56. The method of claim 52, wherein the compound of formula 1c is reduced to 1d by treating with sodium borohydride in the presence of cerium (III) salt.
 57. The method of claim 53, wherein the compound of formula 1c is reduced to 1d by treating with sodium borohydride in the presence of cerium (III) salt.
 58. The method of claim 50, wherein step (iii) comprises esterifying a compound of formula 1d wherein R^(1d) is H, oxidizing and dehydrogenating.
 59. The method of claim 51, wherein step (iii) comprises esterifying a compound of formula 1d wherein R^(1d) is H, oxidizing and dehydrogenating.
 60. The method of claim 52, wherein step (iii) comprises esterifying a compound of formula 1d wherein R^(1d) is H, oxidizing and dehydrogenating.
 61. The method of claim 53, wherein step (iii) comprises esterifying a compound of formula 1d wherein R^(1d) is H, oxidizing and dehydrogenating.
 62. The method of claim 54, wherein step (iii) comprises esterifying a compound of formula 1d wherein R^(1d) is H, oxidizing and dehydrogenating.
 63. (Cancelled). 